Microsoft PowerPoint - 蛋白質體a.ppt

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1 蛋白質體學及應用中山大學生醫所黃弘文

2 內容介紹何謂蛋白質體學蛋白質體學研究方法蛋白質體學應用實驗技術

3 何謂蛋白質體學蛋白質體學與傳統生化學傳統研究蛋白質的科學生物化學酵素學等

4 蛋白質的構造細胞的功能主要由蛋白質執行代謝生化反應繁殖蛋白質為一長鏈的胺基酸聚合物, 鏈長由數十至數千生物體約有 20 種胺基酸

5 胺基酸構造 Functional group: 1.-NH 3 2.-COOH 3.-R ( 羧基 ) 4. 鏡像結構

6 20 種胺基酸 : 依 R group 分類 : 1. 非極性 2. 極性 3. 正電 4. 負電

7 蛋白質鏈方向性 (N C )

8 蛋白質四元結構

9 傳統研究蛋白質的方法蛋白質濃度測定蛋白質 ( 酵素 ) 反應測定蛋白質分離純化蛋白質定序

10 各種蛋白質定量法 : H 206 nm (carbonyl) 280 nm (aromatic) UV Absorbance Special Binding Groups (heme) OH Tyr Lys Cu 2+ Biuret Method... phsophomolybdicphosphotungstate Coomassie Blue G (carbonyl) Lowry Method NH N=N NH NH 3 HN C OH SO - 3 NH SO - C 3 2 SO - C 3 N=N NH C Fe O C OH SO - 3 C C C H H C SO - R H O CR 3 N N O C C C N C C R O N C R C C C H + R R C - O (metal) C N H O H Active O H N H Site N N C C R C C H 2 N H O C O N C C R O H O R SO 3 - Arg NH 2 尿素 urea

.. phsophomolybdicphosphotungstate Coomassie Blue G (carbonyl) Lowry Method NH 2 + + N=N NH NH 3 HN C OH SO - 3 NH SO

11 Bradford Method: Coomassie Brilliant Blue G-250 CBG 是一種指示劑 470 nm 595 nm 加入蛋白質酸性環境下呈茶色與蛋白質結合變藍色

12 酵素反應及偵測方法 : 移除生成物並轉成可測量者使用大量反應物 10 x K m 可能有回饋抑制 - 酵素 + NAD 直接測量 NADH - 耦合反應反應物生成物能否直接測量? 把生成物再轉換成可被測量的生成物

13 蛋白質的分離電泳 (electrophoresis) 色層分析法 (column chromatography)

14 電泳形式的演進 : - 濾紙電泳 Cellulose Protein Denatured 薄層電泳 (TLE) Cellulose acetate Partial Denatured + 膠體電泳 Starch Gel

15 兩種主要蛋白質染色法 : 金屬銀沉澱 + NH 2 Glu NH C C C (Cys) H Ammoniacal silver Coomassie + Blue R Ag H 3N...Ag...NH 3 Lys Arg NH + - SO N=N NH 2 H 2N... Ag NH 3 HN C OH SO - 還 3 - NH C SO Glutaraldehyde 原 SO C 3 C N=N NH Lys C C O OH SO - 3 C C H H C - RC C SO H O R 3 N N O C C C N C C R O N C R C C C H + R R C - O C N H O H O H N H N N C C R C C H O C O N C R O H R

.. Ag NH 3 HN C OH SO - 還 3 - NH C SO Glutaraldehyde 3 2 - 原 SO C 3 C N=N NH Lys C C O OH SO -

16 電泳膠體蛋白質的溶離 : 直接挖出膠體進行溶離或定序 Little Blue Tank ISCO: Little Blue Tank Concentrator

17 電泳槽及相關設備 : 轉印三明治轉印槽電泳槽鑄膠器供電器

18 轉印及免疫染色流程 : A 轉印三明治 B 免疫染色流程及結果塑膠夾板 Filter Paper 轉印抗體 HRP - + Gel Nitrocellulose 海綿墊電泳膠體 Coomassie Blue Staining 轉印紙 Ponceau Staining 抗體偵測呈色

19 SDS-PAGE Disc-PAGE 澱粉磷解染色方法比較 : 膠體過濾法各分劃免疫轉印硝酸銀活性染色

20 蛋白質體 (Proteomics) 二維電泳 (2-Dimensional protein gel electrophoresis) and HPLC were long used in protein separation. 近年生物質譜技術快速發展 (development of mass spectrometry in recent years, ~2000 AD) 2002 Nobel prize: John B. Fenn (Virginia Commonwealth Univ.) Koichi Tanaka (Shimadzu Corporation, Japen) Soft desorption ionization( 軟式脫附離子化 ) methods for mass spectrometric analysis. proteins annotation( 蛋白質註解 ).

近年生物質譜技術快速發展 (development of mass spectrometry in recent years, ~2000 AD) 2002 Nobel prize:

21 Functional Genomics (or Phenomics) The OMICS based sciences! 基因體學轉譯體學蛋白體學代謝體學系統生物學

22 Summary of the systems -ome--- Compete set. -ics --- the utility for analyzing these domains. 功能基因體學 (Functional genomics) : hierachical approaches for studying the functional analysis of novel genes

23 Defining proteomics Functional proteomics: 利用 2DE 及 MS 研究蛋白表現二維電泳 (2DE): two dimensional gel analysis, 通常指 : sample first run a 等電位電泳 IEF (iso( iso- electrofocusing) ) electrophoresis. 再以 SDS PAGE 分離蛋白的方法. 質譜 (MS): mass spectrometry, basically, a method to accurate determine molecule weight.

24 比較蛋白質體學與蛋白質化學 Differences between protein chemistry and proteomics Protein chemistry Proteomics Individual proteins complex mixtures Complete sequence analysis partial sequence analysis Emphasis on structure and function emphasis on identification by database matching Structural biology systems biology Conventional ideas: Using physical biochemistry or mechanistic enzymology, study one protein or multisubunit protein complex at a time. Proteomic ideas: Study multiprotein systems, in which the focus is on the interplay of multiple, distinct proteins in their roles as part of a larger system or network. Proteomics: to characterize the behavior of the system

25 蛋白質體學因應基因體學而來 : Proteomics and Genomics Genomics Genome sequencing Complete DNA sequences from various organisms Bioinformatics Tools Tools for sequence comparison Identification of homologues Tools Tools for sequence interpretation Elucidation of coding sequences / protein functions Gene expression (protein) profiling Transcriptomics Differential expression of mrnas (MicroArray( MicroArray) 基因晶片已知 mrna 表現, 為何還須知道蛋白質表現?

26 DNA 基因基因體學 (Genomics) 轉錄 mrna 功能性基因 Microarray X (...) t-rna t-rna 核糖體 (...) 轉譯蛋白質轉譯後修釋蛋白質體學 (Proteomics) 功能性蛋白質 ( 生化反應 ) Proteomic 2001 Proteomics Group, Institute of Biological Chemistry, Academia Sinica X CHO PO 4 醣質化磷酸化 1. 蛋白質體學與基因體學兩者相互互補 ( 如表現量 ) 但不一定相同 2. 蛋白質體學能發現在基因體學無法完成得到的結果 ( 如醣質化及磷酸化 ) X

27 蛋白質體的四個關鍵技術 : 1. Sample preparation, 樣本處理 2. Protein information, 蛋白質資訊 3. Protein Identification and quantification, 蛋白質註解及定量 4. Cell mapping: targeted proteomics (study of protein-protein interactions, posttranslational modifications (PTMs), cellular localization, etc.).

28 蛋白質體目前瓶頸 : Protein determination range 不同蛋白質表現量差異很大 :~ e.g., 10 copies/cell for transcription factor, up to 10 6 copies/cell for abundant molecules. 2D gels, which usually separate ~1,500 spots, present most abundant proteins if a crude protein mixture is used.

29 Protein Copy number and cell quantity: 無法測到低表現蛋白質 Detection in Silver stain range and coomassie stain range.

30 蛋白質二維電泳 2-D D gel electrophoresis 1st D - Isoelectric focusing ( 聚膠電泳 ) - separation on basis of pi 2nd D - SDS gel electrophoresis (SDS 電泳 ) - separation on basis of mass Isoelectric focusing - separation on basis of pi - ph gradient - migrate to their pi and stabilize ph gradient

31 1st -Dimensional gel Electrophoresis 1st PAGE precast gels: 提供高解析及高範圍生物材料分離 ( denatured proteins, polypeptides or nucleotides) from 2,000,000 to 5,000 Da.

32 Polyacrylamide gel electrophoresis Acrylamide : CH2 = CH - CO NH2 N,N' methylene bis acrylamide: CH2 = CH CO NH CH2 NH CO CH = CH2 Polymerization induced by free radicals produced from ammonium persulfate in presence of TEMED (N, N, N', N' tetra methylethylendiamine) TEMED causes free radicals to form from persulfate

33 利用起始劑自由基反應聚合 : Chemical: TEMED and APS. Photochemical: riboflavin-5 - phosphate, or methylene blue. 電泳膠孔徑 ( 由單體濃度及比例決定 ): %, and ratio: acrylamide and Bis-acrylamide Polymerization temp. Gel additives: Urea (smaller pore size) Polyethylene glycol: macroporous.

35 Isoelectric point (pi) Separation by charge: Stable ph gradient Low ph: Protein is positively charged High ph: protein is negatively charged At the isolectric point the protein has no net charge and therefore no longer migrates in the electric field.

36 ph 值的選擇 ph 梯度差越小, 解析度越高. 根據不同的樣品選擇不同 ph 梯度, e.g., The known extreme pi : acidic glycoprotein of the chimpanzee: pi=1.8, Lysozyme from human placenta: pi=11.7. The most used pi range is ph=3 ~ 10.

37 IEF Ideal Lysis Buffer ( 標準配方 ) 8M urea (or 7M urea + 2M thiourea in the case of fractions containing hydrophobic proteins) 2-4% CHAPS 1% IPG buffer (appropriate buffer for ph range to be analysed) 2 mg/ml DTT

38 Factors which may affect pi - amino acid composition - phosphorylation - glycosylation - other post-translational translational modifications

39 2 nd -Dimensional gel Electrophoresis 第二維電泳通常以 SDS PAGE 進行. 傳統 SDS PAGE 須使用聚膠電泳 (stacking gel) 及分離電泳 (separating gel) 合併使用通常二維電泳中, 因為第一維已聚膠, 不使用 stacking gel.

40 SDS (sodium dodecyl sulfate) 極性頭部非極性尾巴切勿吸入 SDS 塵埃以免引起呼吸困難

41 SDS 在蛋白質表面均勻附上一層負電 : SDS 原態蛋白質 boiling 變性蛋白質成一線狀分子

42 Fig 2.3 Kohlrausch boundary, a limited high-voltage gradient Stacking gel: ph6.8, resolving gel: ph8.8, electrode buffer, ph8.3 (Tris-glycine) Sampel(P1-P5) running between two solution: Cl-(leading), and trailing (Gly-) Laemmli system

43 單元體分子量的測定 : SDS-PAGE Mol mass kda kda kda Migration (R f ) Pharmacia: Molecular Markers for electrophoresis

44 Multiphor II IEF System Perform first- and second-dimension dimension separations with the same system. Run up to 12 IPG strips (7-24 cm) simultaneously. With DryStrip Reswelling Tray, samples can be loaded during IPG strip rehydration by including them in the rehydration buffer.

45 Step 1. Rehydration Use DryStrip Reswelling Tray. Step 2. IEF Use the parallel grooves Cap loading

46 Ettan IPGphor IEF System Accommodates all lengths of Immobline DryStrip gels (7, 11, 13, 18, and 24 cm) and can run 12 gels simultaneously. 8,000 V Max

47 BioRad system Isoelectric focusing (IEF) Pre-cast IPG strips Variable ph range (e.g , 3 4-7, , 6-12, 6 4-5) 4 Variable strip length (7, 11, 17, 24 cm) Optimised protein loading 10,000V Max

48 IPGphor ceramic strip holders are available in five lengths: 7, 11, 13, 18, and 24 cm.

49 IPG strips are rehydrated with IPG Buffer in regular strip holders. The sample can be added to the buffer for efficient loading during rehydration.

50 With movable electrodes and sample cup, the Cup Loading Strip Holder accommodates IPG strips up to 24 cm long.

51 1. Apply rehydration solution. 2. Remove protective film from 3.Drystrip in strip holder DryStrip gel. 4. Apply Cover oil 5. Cover on strip holder. 6. Place assembled strip holder on Ettan IPGphor platform.

52 TECHNICAL SPECIFICATIONS Instrument Voltage Current Platform temperature IPG strip lengths Number of strips Reswelling Strip holder material Sample application Software features Ports Operating temperature Relative humidity Line voltage Maximum power Dimensions (H W D) Weight Safety features Safety certifications Integrated Peltier cooling and high-voltage power supply VDC ma C 7, 11, 13, 18, or 24 cm Immobiline DryStrip Gels 12 maximum In strip holder or reswelling tray when using Cup Loading Strip Holder Base aluminum oxide Cover clear acrylic During rehydration or after rehydration when using Cup Loading Strip Holder 10 protocols, 9 voltage ramps or steps each; set rehydration delay; set maximum current limit; set temperature Serial port (RS232) C 0 90% non-condensing VAC 100 W cm 6.8 kg Automatic voltage shut-off when safety lid is opened CE 73/23/EEC (LV directive); UL3101-1; 1; CSA

53 Multiphor II and ExcelGel System Perform second-dimension dimension separations using convenient precast gels and or 24 cm IPG strips. Choose from ExcelGel precast SDS-polyacrylamide gels available as 12.5% homogeneous and 12-14% 14% gradient gels. Prepare multiple 2-D 2 D maps on a single gel using three 7 cm or two 11 cm IPG strips side-by by-side. Configure Multiphor II with MultiTemp III Thermostatic Circulator and the programmable EPS 1001 Power Supply for reproducible running conditions.

54 Hoefer SE 600 Ruby Vertical System Perform second-dimension dimension separations using 7 or 13 cm IPG strips. Prepare multiple 2-D 2 D maps on a single gel using two 7 cm IPG strips side-by by-side. Run up to four gels simultaneously and perform eight second-dimension dimension separations at once. Configure SE 600 with MultiTemp III Thermostatic Circulator and the programmable EPS 601 Power Supply for reproducible running conditions.

55 影像分析 Complex protein patterns in 2D electrophoresis gels are captured in digital format to allow accurate, reproducible and quantifiable comparisons. Image Master (Melanie) (Pharmacia) PDQuest (BioRad)

56 Image Scanner ImageScanner has an extremely broad optical density range to give the high resolution required for reliable quantification of Coomassie and silver stained gels. Fast scanning and image acquisition rates ensure short scan times.

57 Typhoon Typhoon unites four-colour fluorescence, filmless autoradiography and chemiluminescence into a single system. Its broad dynamic range ensures accurate results at the first attempt. Highly sensitive optics facilitate extremely low limits of fluorescence detection and enable direct chemiluminescence imaging without intermediate exposures to film or screens.

58 2-DE Sample prep. Protein 蛋白質體分析流程 Rehydration Mass IEF Protein identify from database SDS-PAGE MALDI-TOF Gel -Image Analysis In-Gel Digestion Reduction Alkylation Extraction Peptide

P3 膠體電泳法

P3 膠體電泳法 Polyacrylamide Gel Electrophoresis 聚丙烯醯胺膠体電泳莊榮輝 3.1 電泳原理帶電分子在電場中能夠移動, 稱作泳動, 泳動的程度稱為泳動率 (mobility).. 泳動率 ~ ( 所外加電壓 mv) ( 分子之淨電荷 ) 分子與介質間之摩擦力上述